This proposal will reveal the structural mechanisms and motifs that enable a tiny viral oncoprotein to target and disrupt multiple critical tumor suppressor pathways. The ultimate goal is to exploit this knowledge to develop novel viral therapies against intractable tumor targets. The evolution of minimal DNA tumor virus' genomes has selected for small viral oncoproteins that hijack critical cellular protein interaction network that are also targeted by mutations in cancer. However, the structural basis for the dominant interactions of small adenovirus oncoproteins has remained elusive, as none of their complete structures have been solved. This represents a fundamental gap in the understanding of Adenovirus biology and the structural principles that enable small viral proteins to 'win'. To address this the structure of an E4-ORF3 dimer at 2.1 was solved. E4-ORF3 is a 13kDa protein that assembles a nuclear polymer network that binds and disrupts the PML, TRIM24, and MRE11/RAD50/NBS1 (MRN) tumor suppressor complexes. In addition, E4-ORF3 induces heterochromatin silencing at p53 target genes and anti-viral genes through an unknown mechanism. In contrast to the archetypal Adenovirus oncoprotein, E1A, E4-ORF3 has a discrete ordered structure and is not a structural homologue of any known cellular polymers or oncogenes. E4-ORF3 forms dimer subunits with a central beta-core that further co-assemble through reciprocal and non-reciprocal exchanges of their C-terminal tails. The higher order assembly of E4-ORF3 is required for creating avidity-driven interactions with PML and an emergent MRN binding interface. This proposed research builds on these studies. Aim 1 will reveal the structure and higher order oligomeric interactions that drive the assembly of wild type E4-ORF3 and are required for its functions in disrupting multiple tumor suppressors to facilitate viral replication. Aim 2 will use the structure of E4-ORF3 as a rational basis to identify new structural motifs that target the PML, MRN and TRIM24 tumor suppressor complexes, which are important therapeutic targets. Discrete E4-ORF3 mutations will be engineered that selectively uncouple its interactions with different tumor suppressor complexes to reveal their respective contributions in viral infection. This will provide a rational basis for the development of novel vral cancer therapies that selectively replicate in tumor cells with particular tumor suppressor pathway mutations. E4-ORF3 dimerization creates a novel binding-cleft that determines the sites of its assembly in the nucleus and is required for silencing p53 target genes. Aim 3 will use a combination of viral engineering and integrative comparative genomics approaches to determine if residues within the cleft target the E4-ORF3 assembly to specific genomic loci where it binds to a motif in H10 and to induce repressive heterochromatin silencing of p53 and anti-viral genes. This will reveal new targets and mechanisms that silence p53 in infection and that could also be disrupted by mutations in cancer.